Ras oncogenes are the most frequently mutated in human cancer. The Ras proteins are small GTPases that control critical cellular pathways for growth, proliferation, and survival, dysregulation of which is implicated extensively in cancer. Of the three Ras genes, mutations in KRAS are the most frequently found in human tumors. There are two splice variants encoded by the KRAS gene: K-Ras4A and K-Ras4B. Much work has been done to characterize Ras biology, but the vast majority of studies have overlooked the K-Ras4A isoform, despite its importance as the original K-Ras viral oncogene. Recent studies have begun to implicate a greater role for K-Ras4A in cancer than previously assumed, but little is still known about the K-Ras4A protein. This proposed work seeks to rectify this by elucidating the cell biology of K- Ras4A with two specific aims. First, we will investigate the targeting signas for K-Ras4A localization and how they are regulated. As membrane association is required for Ras signaling, determining how K-Ras4A is trafficked within the cell is critical in understanding how it exerts its signaling effects. We hypothesize that K-Ras4A is trafficked to the plasma membrane by combined palmitoylation and polybasic interactions, and will test this with live cell imaging studies. Second, we will explore the role that K-Ras4A plays in mediating oncogenesis. We will examine in human cancer cell lines the relative expression levels and dependence on K-Ras4A for survival and tumorigenesis, and study the relative signaling outputs of the two K-Ras isoforms. Our findings from these studies will provide a better understanding of this elusive isoform and contribute to our knowledge of Ras biology. This work will also have significant implications for the design and development of drugs against cancers driven by K-Ras. PUBLIC HEALTH RELEVANCE: K-Ras is the oncogene most frequently mutated in human cancer, and is implicated in more than 30% of human tumors. Understanding the importance and role of the splice isoform K- Ras4A is thus highly significant for the rational design of drugs combating cancers driven by mutations in K-Ras.